From Surface Colonies to Internal Contamination: A Comprehensive Investigation of Alternaria alternata Growth, Toxinogenesis, and Mycotoxin Migration Dynamics in Cherry Tomato Fruit Matrix

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled "From surface colonies to internal contamination: A comprehensive investigation of Alternaria alternata growth, toxinogenesis, and mycotoxin migration dynamics in cherry tomato fruit matrix" reports an interesting research activity focused on the ability of Alternaria alternata and its toxic metabolites to colonize and migrate in tomato fruit. 

The manuscript is well-written, and the experimental design and methodology are well-presented. I only recommend modifying the title, as it is very long and unattractive, and removing Figure 1, which is very redundant. Anyone working on Alternaria is familiar with the morphology of Alternaria on MEA, PDA, etc. 

At the same time, it is appropriate to make the paragraph 2.1 more concise, as the different growth capacities on these media are also well documented.

Line 88 Growth patterns and toxin production of Alternaria alternata in different in vitro media rephrase "Fungal growth and mycotoxin production in different media"

Line 93 add full name and composition of the media; the name of the strain and remove identified by PCR.

Line 147 rephrase the title

Line 411 remove "such as"

Author Response

We are really appreciated and thank you for your participation and kind remarks. (Note: Please turn off track changes to follow the line guiding.)

Comment 1:The manuscript is well-written, and the experimental design and methodology are well-presented. I only recommend modifying the title, as it is very long and unattractive, and removing Figure 1, which is very redundant. Anyone working on Alternaria is familiar with the morphology of Alternaria on MEA, PDA, etc.  At the same time, it is appropriate to make the paragraph 2.1 more concise, as the different growth capacities on these media are also well documented. 

Respond: We thank you sincerely for your suggestion regarding the title. We agree that it is indeed quite long. However, for now, we prefer to keep it as it provides a complete representation of the content presented in the article. During the final revision stage, we will take the opportunity to reflect further and propose a more concise and solid title. In addition, all figures have been revised. Figure 1 has been modified to present representative quantitative data, and we believe that including mold morphology alongside these data (Figure 1a) now provides complementary information that supports the revised presentation. The corresponding text has been revised accordingly. We also believe that this combined representation may be helpful for readers who are less familiar with Alternaria, thus developping an attraction for the field (Line 97 to 131).

 

Comment 2: - Line 88 Growth patterns and toxin production of Alternaria alternata in different in vitro media rephrase "Fungal growth and mycotoxin production in different media" ; - Line 93 add full name and composition of the media; the name of the strain and remove identified by PCR; - Line 147 rephrase the title; - Line 411 remove "such as" 

Respond: It’s been corrected, thank you for all these suggestions. (Line 97, Line 113-114, Line 155, Line 540, respectively) 

Reviewer 2 Report

Comments and Suggestions for Authors

The current study was an very interesting report about alternaria alternata growth, toxinogenisis and mycotoxin migration dynamics in cherry tomato fruit matrix. The study design was good and the results were well presented. More impotant, the findings of this study benefits consumers and will gain wide attention. Thus, I recommend publication of this paper in Toxins after only minor revisions.

1. for all fugures, the authors should add figure caption to describe Layer S1-S4.
2. for mycotoxin extraction and quantification, the Limit of detection and the limit of quantification should be mentioned.
3. P644-645, no meed to add reference for Kruskal-Wallis test, as this statistical method has been widely used.
4. For the strain identification, the NCBI accession number of the DNA sequences should be added.

Author Response

We truly appreciate your participation and thank you for all your suggestions. (Note: Please turn off track changes to follow the line guiding.)

Comment 1 and 2: for all figures, the authors should add figure caption to describe Layer S1-S4; for mycotoxin extraction and quantification, the Limit of detection and the limit of quantification should be mentioned.

Respond: We have revised the presentation of all figures in the new manuscript to improve visual clarity and facilitate interpretation, in which, all the data below LOQ have been excluded. The description of figure layer are now represented in table 2 (Line 191-193). We would also like to clarify that, in the new Figure 5 (line 335), quantitative values differ from those previously reported in the old version of the manuscript. This difference results from a calculation error in the earlier version, in which the dilution factor was inadvertently omitted. Although absolute values have changed, the trends and patterns remain unchanged. The heatmap representation allows for clearer visualization and interpretation of degradation dynamics for that patterns.

 

Comment 3: P644-645, no meed to add reference for Kruskal-Wallis test, as this statistical method has been widely used.

Respond: Agree. It's corrected. (Line 813)

 

Comment 4: For the strain identification, the NCBI accession number of the DNA sequences should be added.

Respond: Thanks you. We have added the identification in the new manuscript (OP758812.1) (Line 694)

Reviewer 3 Report

Comments and Suggestions for Authors

This paper describes an investigation of Alternaria alternata growth and mycotoxin migration dynamics in cherry tomato fruit. This paper focus on a scientific relevant problem about the presence of toxic mycotoxin in cherry tomato fruit food. The research seems to be well design and experimentally executed. The following minor questions should be optimized:

• The standard deviations of the experimental measurements must be shown - only the average values are presented in the text.

Author Response

We sincerely thank you for your participation and truly appreciate your kind review. (Note: Please turn off track changes to follow the line guiding.)

Comment: The standard deviations of the experimental measurements must be shown - only the average values are presented in the text.

Respond: We are very thankful for this suggestion. We have revised the presentation of all figures, particularly those related to the in vivo model (Figure 3) (Line 236) and the spiking model (Figure 5) (Line 335), to improve visual clarity and facilitate interpretation. 

Plus: We would also like to clarify that, in Figure 5, quantitative values differ from those previously reported in the old manuscript. This difference results from a calculation error in the earlier version, in which the dilution factor was inadvertently omitted. Although absolute values have changed, the trends and patterns remain unchanged. The heatmap representation allows for clearer visualization and interpretation of degradation dynamics for that patterns. The corresponding text has also been revised accordingly in the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you very much for considering me as a reviewer for the manuscript entitled “From surface colonies to internal contamination: A comprehensive investigation of Alternaria alternata growth, toxinogenesis, and mycotoxin migration dynamics in cherry tomato fruit matrix ”. The manuscript is very interesting and well written. I agree that research aiming to understand toxin distribution and reduce food waste is highly relevant. Nonetheless, the manuscript is rather long and covers many topics. I would suggest that the manuscript be shortened, that some figures be removed, and that certain experiments potentially be omitted. For example, the in vitro test does not seem to add novel information.

Below is a detailed and non-exhaustive list of suggestions for the authors to address in order to improve the manuscript:

Introduction: It is not clear from the introduction that an in vitro test was performed, nor what its purpose was. This should either be clearly stated in the introduction or the in vitro experiment should be removed.

Line 52: avoid repetition of the word “among”   

Line 93: Why was this strain selected? Is it part of a strain collection? What makes this strain particularly relevant compared to others?

Line 586: why all the sections were pooled? Sections from duplicates? This is not clear and should be clarified in the text

Quantification of mycotoxins: Why was the internal standard added at the end of the procedure? In this case, recovery must be taken into account; otherwise, accurate quantification cannot be ensured. Was quantification performed using matrix-matched calibration curves, as established in regulations? How was quantification performed and within which concentration range? More detailed information is required.

How was it possible to quantify toxins on different days from the same medium or tomato? Were replicates performed? This is not clearly described and should be clarified.

Degradation of mycotoxins: Information or hypotheses regarding into what the toxins may have degraded, even if only theoretical, would be valuable to include in the discussion. It is also possible that the toxins undergo modification or bind to the matrix, and this should be discussed.

Figure 12: If TEA is detected, how can the tomato still be considered consumable? It should be clarified in the table that the contaminated area must be removed. Even if toxin levels are low, I would not recommend consumption, as this contributes to overall exposure; several risk assessments already show that exposure to Alternaria toxins is high based solely on processed food consumption. Recommending additional exposure does not seem appropriate. Moreover, this study was conducted using only one strain, so how can it be assumed that all strains are equally toxigenic? There are Alternaria species with higher or lower toxigenic potential capable of infecting tomato fruit. Additionally, how can consumers identify Alternaria contamination? For the recommendations, I suggest either performing a risk assessment to evaluate how much tomato could be safely consumed or removing these recommendations altogether.

Some of the figures should be moved to supplementary material.

Figure 3 needs some modification. Either group the toxins by day, or the days for each toxin. I n its current form, it is very long and difficult to interpret.

Figure 6: the scale needs to be adapted. Keep the same scale for all the graphs, but a max of 100000 should be fine to observe the differences between the sections.

Author Response

We truly appreciate the time and effort you dedicated to carefully reading our manuscript and providing such detailed comments, and we sincerely thank you! We have addressed all the points you raised and believe that the revised version has been significantly strengthened as a result. (Note: Please turn off track changes to follow the line guiding.)

 

Comment 1: Introduction: It is not clear from the introduction that an in vitro test was performed, nor what its purpose was. This should either be clearly stated in the introduction or the in vitro experiment should be removed.

Respond: We agree that the rationale for the in vitro experiment was not sufficiently explicit in the Introduction. The in vitro assay was conducted as a preliminary and complementary step to (i) characterize the intrinsic toxigenic potential of the A. alternata strain under controlled conditions, (ii) confirm the production of the three Alternaria toxins (TeA, AOH, and AME) quantified throughout the study, and (iii) provide a baseline reference to support the interpretation of toxin production observed in the tomato matrix, given that culture medium composition can influence fungal secondary metabolism. A clarifying sentence has been added, but at the begining of the in vitro’s section result for a better narrative (line 109-112).

 

Comment 2, 3: Line 52: avoid repetition of the word “among”; Line 93: Why was this strain selected? Is it part of a strain collection? What makes this strain particularly relevant compared to others?

Respond: We have revised the Intro to improve clarity and grammar. Regarding the strain selection (line 61), the revised Intro now states that among mycotoxin-producing fungal genera affecting tomato fruits, Alternaria species are the most frequently observed, with Alternaria alternata being the most commonly isolated species, supported by newly added references. Also, The strain used in this study is part of our isolate collection established within the framework of the MYNION project, as described in Section 5.1 (line 645). As the project is still ongoing, more detailed information on the broader strain collection cannot yet be provided. We are sorry for this inconvenient!

 

Comment 4: Line 586: why all the sections were pooled? Sections from duplicates? This is not clear and should be clarified in the text

Respond: For each experimental condition, 6 independent cherry tomatoes were sectioned into defined sections. Corresponding sections from the 6 fruits (for ex, all S1 sections, all S2 sections, ...) were pooled together in a single tube. From each pooled section sample, triplicate analytical measurements were then performed. This is what we implied in « 3 repeated experiments for 6 fruits » (Line 753-755)

 

Comment 5: Quantification of mycotoxins: Why was the internal standard added at the end of the procedure? In this case, recovery must be taken into account; otherwise, accurate quantification cannot be ensured. Was quantification performed using matrix-matched calibration curves, as established in regulations? How was quantification performed and within which concentration range? More detailed information is required.

Respond:  Our method follows the routine reported by Dole et al. (Routine LC-MS/MS method for quantifying Alternaria toxins in tomatoes at harvest stage and during processing) (Line 768-770), in which :

• Calibration curves are constructed using the ratio of toxin peak area to internal standard peak area for AOH and AME (and other toxins), while TeA is quantified from its peak area.
• Calibration is expressed with linear ranges of 2.5-50 ng/mL for AOH, 2.5-25 ng/mL for AME, and 5-100 ng/mL for TeA, and R² > 0.9900.

In the revised manuscript, values below the LOQ are now reported as non-detected to improve data clarity and avoid more confused.

 

Comment 6: How was it possible to quantify toxins on different days from the same medium or tomato? Were replicates performed? This is not clearly described and should be clarified.

Respond: 

-For the in vitro experiment, all culture media were prepared and inoculated at day 0. For each sampling time point, 3 independant samples were collected and analyzed, as described in Section 5.4.

-For the tomato in vivo experiments, a similar experimental design was applied, but using a larger number of units. Specifically, 6 independent tomatoes were used for each sampling time point. Sampling was performed using the pooling strategy described above, whereby corresponding sections from the 6 fruits were pooled prior to analysis. This procedure and the associated replication strategy are detailed in Section 5.5 (line 735).

 

Comment 7: Degradation of mycotoxins: Information or hypotheses regarding into what the toxins may have degraded, even if only theoretical, would be valuable to include in the discussion. It is also possible that the toxins undergo modification or bind to the matrix, and this should be discussed.

Respond: We are very thankful for this suggestion. A new section presenting a theoretical discussion has been added to the revised manuscript to address the potential mechanism degradation (Line 496-530)

 

Comment 8: Figure 12: If TEA is detected, how can the tomato still be considered consumable? It should be clarified in the table that the contaminated area must be removed. Even if toxin levels are low, I would not recommend consumption, as this contributes to overall exposure; several risk assessments already show that exposure to Alternaria toxins is high based solely on processed food consumption. Recommending additional exposure does not seem appropriate. Moreover, this study was conducted using only one strain, so how can it be assumed that all strains are equally toxigenic? There are Alternaria species with higher or lower toxigenic potential capable of infecting tomato fruit. Additionally, how can consumers identify Alternaria contamination? For the recommendations, I suggest either performing a risk assessment to evaluate how much tomato could be safely consumed or removing these recommendations altogether.

Respond: We are very thankful for this valuable guidance! The recommendation table has been fully revised and is now presented as Figure 6 (line 554-599). In this new figure, worst case scenarios derived from the study data are considered, linking toxin concentrations to levels of visible contamination. The recommendations are now framed using three precautionary categories (recommended, precaution, and discard), based on the Threshold of Toxicological Concern (TTC), rather than promoting consumption. With this revised version, we  aims to illustrate a conservative, risk-oriented framework rather than to encourage intake of contaminated products.

 

Comment 9:  Some of the figures should be moved to supplementary material.

• Figure 3 needs some modification. Either group the toxins by day, or the days for each toxin. I n its current form, it is very long and difficult to interpret.
• Figure 6: the scale needs to be adapted. Keep the same scale for all the graphs, but a max of 100000 should be fine to observe the differences between the sections.

Respond: We have reworked all figures in the revised manuscript to improve visual clarity and data interpretation. The data previously shown in old Figure 3 have now been included in Table 1 (line 132), where toxin concentrations are presented as min–max values derived from 3 replicates. For old Figure 6, the data are now presented in the revised Figure 3a (line 236), allowing simultaneous comparison between fungal DNA concentration and toxin concentration across the different sections in the same figure 3 (line 236). In addition, all values below LOQ have been excluded from the figures to provide a clearer and more interpretable graphical representation.

 

Plus: We would also like to clarify that, in the revised representation of the spiking model data (Figure 5) (line 335), quantitative values differ from those previously reported. This difference results from a calculation error in the earlier version, in which the dilution factor was inadvertently omitted. Although absolute values have changed, the trends and patterns remain unchanged. In addition, the heatmap representation allows for clearer visualization and interpretation of degradation dynamics. The corresponding text has also been revised accordingly in the manuscript.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed all my comments and the manuscript has substantially improved. I have no futher comments. 

Author Response

We are sincerely grateful for your thoughtful and constructive comment. Your feedback was truly helpful in improving the latest version of our manuscript. We wish you all the best.